PROJECT SUMMARY Adoptive cell transfer introduces engineered tumor-targeting cytotoxic T lymphocytes (CTLs) to patients. While this immunotherapy is effective against hematologic malignancies, it is ineffective against solid tumors due in part to the immunosuppressive microenvironment. The tumor microenvironment presents many challenges to CTL energy production including oxygen and glucose depleted environment and tumor expression of inhibitory ligands that limit nutrient uptake by T cells. These conditions render CTLs hypo-responsive. Nave T cells rely on OxPhos for energy. Following activation, the highly proliferative effector T cell undergoes metabolic remodeling and shifts reliance from OxPhos to glycolysis. As such, T cells are considered metabolically plastic and massive metabolic alterations are a normal part of T cell development. However, how metabolic alterations impact cell fate and function at their target tissue sites remain unknown. This leads us to our central hypothesis that boosting metabolism at the tumor site can prevent CTL dysfunction and improve adoptive cell transfer immunotherapy outcomes. Unfortunately, to date there has been no means of testing this hypothesis directly. Metabolic reprogramming is commonly studied through the global administration of drugs that lack target selectivity. We aim to circumvent this limitation through our novel optogenetic systems that allow us to directly modulate T cell metabolism. We will; (1) investigate the impact of the mitochondrial membrane potential on CD8+ T cell effector functions, (2) regulate local cytokine signals to control T cell metabolic programs in the tumor microenvironment, and (3) develop deep tissue immunomodulation approaches. The completion of the proposed study will enable us to gain a comprehensive analysis of immunity that can provide new insight into how T cells interact with the tumor microenvironment.